Mucus is a viscous gel, the properties of which are dependent on a variety of factors. Mucus is primarily made up of a mixture of variable amounts of mucous glycoproteins, water, low molecular weight ions, proteins and lipids. These components interact in a number of ways and these interactions create the three-dimensional structure of the gel and determine the gel's viscosity and elasticity.
Mucin is the principle polymeric component of the mucus gel and consists of a peptide backbone with glycosylated and non-glycosylated domains and oligosaccharide chains. The presence of sulphated and sialic terminals makes the molecule highly polyanionic. The mucins form a polydisperse group of densely charged linear polymers, some of which are up to 6 μm in length, with random tangles. The theological properties are mainly dependent on the tangle density, which in turn is determined by the degree of mucus hydration and mucin molecule length. The necrotic activated neutrophils release large amounts of DNA, actin and proteins which also polymerise and interact with mucin. This process considerably increases the tangle density to form highly viscoelastic mucus gels.
A variety of different types of bonds within airway mucus affect the chemical and physical properties of the mucus, such as viscoelasticity. Disulphide bonds are covalent bonds which link glycoprotein subunits into the large, extended macromolecular chains known as mucins. Cross-links form between adjacent mucin polymers, probably as a result of their large size. The sugar units, which make up the oligosaccharide side-chains and account for about 80% of the mucin weight, form hydrogen bonds with complimentary units on neighbouring mucins. Although each individual bond is weak and readily dissociates, there are very large numbers of bond sites, making this a significant type of bonding within the mucus. In addition, mucins are also ionized, containing both positively charged amino acid residues as well as negatively charged sugar units, principally sialic acid and sulphated residues. The degree of mucin ionisation may actually increase in airway disease. For example, in cystic fibrosis (CF) the proportion of sulphated residues is further elevated because of alterations in glycosyl transferase activities within the Golgi apparatus. The ionic interactions between fixed negative charges result in a stiffer, more extended macromolecular conformation, effectively increasing the polymer size and adding to the numbers of entanglements. Finally, in airway diseases characterized by infection and inflammation, such as CF, high molecular weight DNA and actin filaments are released by dying leukocytes, and exopolysaccharides are secreted by bacteria. These add further bonding and bulk to the mucus.
Mucus is a critical component of the primary defence mechanism of the respiratory tract, trapping inhaled particulate and microbial material for removal via the mucociliary system. However, when this mechanism fails to clear sufficiently, mucus accumulates and must be coughed up as sputum, otherwise it is retained in the respiratory tract and can encourage the colonisation by microorganisms which may lead to chronic lung inflammation and obstruction.
Retention of the mucus in the respiratory tract presents a particular problem as it not only obstructs the airways but also facilitates infection and promotes a self-perpetuating cycle of infection and inflammation. Pathological agents such as bacteria (e.g. Pseudomonas aeruginosa) are often able to establish colonies within the mucus.
Problems tend to arise when the initial bacterial infection stimulates neutrophil chemotaxis, but the neutrophils are unable to effectively clear. Defective neutrophil apoptosis and impaired phagocytosis are key factors in the pathogenesis of lung disease in CF. Neutrophil proteases and oxidants are released during the process and these have a number of effects. They cause both cellular damage and impairment of ciliary movement. They are also potent secretagogues and actually enhance further mucus secretion. The proteases also cleave anti-proteases and cell surface markers, further impairing the host defence mechanisms. Thus, the cycle is perpetuated as these effects further impair mucus clearance at the same time as increasing mucus secretion, encouraging bacterial stasis and promoting airway inflammation. Therefore, the failure of the neutrophils to clear the original infection actually leads to a rapid deterioration of the situation and the process accounts for much of the morbidity and mortality observed in patients with CF.
There are two main causes of mucus retention. The first is airway mucus hypersecretion, where the body produces and secretes elevated levels of mucus and the mucociliary system is unable to cope with and clear the large amounts of mucus quickly enough. The second cause is where the mucus has abnormal viscoelasticity. Where the mucus has an unusually high viscoelasticity, it is much more difficult for the mucociliary system to move the mucus and clear it from the airways.
Agents which affect the mucus in a way that assists clearance have traditionally been referred to as “mucolytics” agents. However, this term may be inaccurate, as may of the agents in question do not exert their effect on the mucus by lysis. Therefore, agents which assist mucus clearance are herein referred to as mucoactive agents.
Classical courses of action taken to treat individuals afflicted with airway hypersecretion and/or abnormal mucus viscoelasticity include antibiotic therapy, administration of bronchiodilators, use of systemic or inhaled corticosteroids, or oral administration of expectorants for liquefaction of the mucus. It is also known to treat the sufferers with aerosol delivered “mucolytic” agents, such as water and hypertonic saline solution. Recombinant human DNase I (rhDNase) has been used to treat CF sufferers. The rhDNase is thought to enzymatically digest the naked DNA released into the airway surface fluid from bacteria, neutrophils, and other cellular debris. It is this DNA which is thought to contribute to the elevated viscoelasticity of the mucus in CF sufferers.
However, these conventional approaches have met with only limited success and there is the need for cheap and effective treatment for mucus retention in the lungs. What is more, it is an aim of the present invention to provide a treatment which will lead to a reduction in the mucus elasticity and viscosity and which will result in improved cough and airway clearance of the mucus and also enable clearance by means of ciliary action.
Agents such as rhDNase, which digests the naked DNA in the mucus, and gelsolin, which digests actin in the mucus, have been shown to affect the elasticity components of the network, as opposed to the viscosity. In model studies, this will tend to improve cough and airway clearance, rather than helping clearance by means of ciliary action.
It has been suggested that agents which disrupt the cross-links in the mucus cause a reduction in both elasticity and viscosity. This is the preferred result, as it will lead to an improvement in ciliary clearance, according to model studies.
Dextrans have been identified as being a potentially useful agent for improving mucus clearance in International Publication No. WO 99/01141. In this patent application, it is suggested, from in vitro models, that dextrans decrease mucus viscoelasticity and increase mucociliary clearability. The dextrans are thought to have this effect by disrupting the hydrogen bonding between mucins within the three-dimensional mucus structure. It is hypothesised that the dextrans compete with the mucin for the hydrogen bonding sites, resulting in the substitution, by dextran carbohydrate moieties, of oligosaccharide moieties linked to high molecular weight mucin peptides that make up the mucus gel. The dextrans used have significantly lower molecular weight and so these new hydrogen bonds are structurally and rheologically ineffective, thus reducing the overall cross-link density within the mucus and this, it is believed, improves mucus clearance by ciliary and cough mechanisms.
In a later patent application, International Publication No. WO 01/15672, it is further suggested that the action of dextrans may be further enhanced by using charged forms. A charged dextran, for example dextran sulphate, is thought to have dual activity. Firstly, it is said to have the effects due to competition for hydrogen bonding sites as discussed above. Secondly, the ionic nature of the charged dextran is thought to have an additional effect, shielding some of the fixed charges along the macromolecular core of the mucin polymer, making it less stiff and reducing the number of entanglement cross-links with neighbouring macromolecules within the mucus and thereby reducing viscoelasticity due to ionic interactions.
In WO 01/15672, it is also suggested that the charged oligosaccharide heparin is not suitable for treating pulmonary diseases such as CF, because it is expensive to produce and, more significantly, because it could potentially have toxic side-effects such as pulmonary haemoptysis, which is bleeding of the tracheobronchial mucosa.
Heparin is a linear polysaccharide which, along with related proteoglycans such as heparan sulphate, is a member of the group of macromolecules referred to as glycosaminoglycans. Owing to their linear anionic polyelectrolyte structure, these macromolecules are involved in various biological processes. While heparin has been used largely for its anticoagulant effects based on its binding to plasma anti-thrombin III, there is evidence that heparin and other glycosaminoglycans also possess various anti-inflammatory and immunoregulatory properties, including the modulation of T-lymphocytes, complement activation, inhibition of neutrophil chemotaxis, smooth muscle growth and reduction of intrinsic DNA viscosity.
Heparin is a heterogeneous mixture of variably sulphated polysaccharide chains with a molecular weight range of 6000 to 30,000 Daltons. Whole or unfractionated heparin (UFH) may be fractionated to give low and high molecular weight fractions, as is well known in the art. Fractionated, low molecular weight heparin (LMWH) has been shown to reduce the viscoelasticity of dog mucus and improve mucociliary clearance on a frog palate model.
The effects of inhaling an aqueous solution of heparin using a nebuliser on bronchial asthma have been the subject of several studies. However, the results of these studies have been inconsistent, possibly because of the difficulty in quantifying the dosages of inhaled heparin reaching the lower respiratory tract.
N-acetyl-L-cysteine, which is also commonly called acetylcysteine or NAC, is a chemical produced by the body that enhances the production of the enzyme glutathione, a powerful antioxidant. NAC is known to be a mucactive agent and is used to help break up the thick mucus often present in people suffering from chronic respiratory ailments. It is available in an oral solution as Mucomyst (trade mark) that can be ingested or aerosolised and inhaled.